Nanoparticle-peptide compositions

ABSTRACT

The present invention provides nanoparticles and compositions comprising such nanoparticles, as well as methods for intracellular delivery of peptides, and methods of producing nanoparticles and related products. The nanoparticles comprise a core comprising a metal and/or a semiconductor atom; and a corona comprising a plurality of ligands covalently linked to the core, wherein at least a first ligand of said plurality comprises a carbohydrate moiety that is covalently linked to the core via a first linker, and wherein at least a second ligand of said plurality comprises a peptide of choice that is covalently linked to the core via a second linker. The second linker comprises a peptide portion and a non-peptide portion, wherein said peptide portion of said second linker comprises the sequence X 1 X 2 Z 1 , wherein: X 1  is an amino acid selected from A and G; X 2  is an amino acid selected from A and G; and Z 1  is an amino acid selected from Y and F.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/EP2012/067561, filed Sep. 7, 2012, which claims benefit of U.S.Provisional Patent Application No. 61/531,745, filed Sep. 7, 2011. Theentire disclosure of each of the aforesaid applications is incorporatedby reference in the present application.

FIELD OF THE INVENTION

The present invention relates to substances and compositions useful inintracellular peptide delivery, in particular nanoparticle-mediateddelivery of peptides. In certain cases, the invention relates tointracellular delivery of epitopic peptides, e.g., to induce a T cellresponse. Intracellular peptide delivery may be employed for therapeuticand/or prophylactic treatment of, in particular, tumours and/orpathogen-mediated disease, such as bacterial, viral or parasiteinfection.

BACKGROUND TO THE INVENTION

A significant challenge for the design and development of peptide-basedtherapy, such as immunotherapy, is the intracellular delivery of thepeptides to intracellular compartments. An example of this is the needto deliver antigen peptides to the antigen processing machinery suchthat the peptides are presented bound to a class I MHC molecule andthereby stimulate a CTL response. Other examples include delivery ofpeptide-based drugs to intracellular drug targets. In particular, freepeptides often display poor uptake by cells.

One approach that has been employed to date, is to deliver peptides to aintracellular location by administering a vector, such as a viralvector, containing a polynucleotide that encodes the peptide of choicesuch the that cell can be transfected with the vector and the peptide isproduced by the cellular translational machinery. A further example of astrategy to deliver a peptide to an intracellular location is describedin Muders et al., 2009, Clin Cancer Res, Vol. 15(12), pp. 4095-4103, inwhich a GIPC-PDZ-targeting peptide was delivered to certain pancreatictumour cells. The peptide was modified by N-terminal myristolation.

However, there remains a need for further methods and compositions forintracellular delivery of peptides, e.g., in order to induce an immuneresponse or for the administration of certain peptide-based drugs. Thepresent invention addresses these and other needs.

WO 2006/037979 describes nanoparticles comprising antigens andadjuvants, and immunogenic structures comprising the nanoparticles.

BRIEF DESCRIPTION OF THE INVENTION

Broadly, the present invention relates to nanoparticle-basedintracellular delivery of peptides. The present inventors have foundthat the nanoparticles of the invention, described herein, which have acorona including a peptide of choice that is covalently attached to thecore of the nanoparticle via particular linkers, are capable of beingtaken up by cells and thereby delivering the peptide of choice to anintracellular target. As described herein, the peptide of choice maythen be released from the nanoparticle, e.g. by specific cleavageprocesses, so as to free the peptide of choice for interaction with anintracellular target. One example of this is the delivery of an epitopicpeptide to an APC for processing and presentation via a class I MHCmolecule such that a CTL response may be generated.

Accordingly, in a first aspect the present invention provides ananoparticle comprising:

-   -   (i) a core comprising a metal and/or a semiconductor atom;    -   (ii) a corona comprising a plurality of ligands covalently        linked to the core, wherein at least a first ligand of said        plurality comprises a carbohydrate moiety that is covalently        linked to the core via a first linker or wherein said first        ligand of said plurality comprises glutathione, and wherein at        least a second ligand of said plurality comprises a peptide of        choice that is covalently linked to the core via a second        linker, said second linker comprising:        -   a peptide portion and a non-peptide portion, wherein said            peptide portion of said second linker comprises the sequence            X₁X₂Z₁, wherein:        -   X₁ is an amino acid selected from A and G;        -   X₂ is an amino acid selected from A and G; and        -   Z₁ is an amino acid selected from Y and F.

In some cases in accordance with the present invention the non-peptideportion of the second linker comprises C2-C15 alkyl and/or C2-C15glycol, for example a thioethyl group or a thiopropyl group.

In some cases in accordance with the present invention the first ligandand/or said second ligand are covalently linked to the core via asulphur-containing group, an amino-containing group, aphosphate-containing group or an oxygen-containing group.

In some cases in accordance with the present invention said peptideportion of said second linker comprises or consists of an amino acidsequence selected from:

-   -   (i) AAY; and    -   (ii) FLAAY (SEQ ID NO: 1).

In certain preferred cases, said second linker is selected from thegroup consisting of:

-   -   (i) HS—(CH₂)₂—CONH-AAY;    -   (ii) HS—(CH₂)₂—CONH-FLAAY (SEQ ID NO: 1);    -   (iii) HS—(CH₂)₃—CONH-AAY;    -   (iv) HS—(CH₂)₃—CONH-FLAAY (SEQ ID NO: 1);    -   (v) HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-AAY; and    -   (vi) HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-FLAAY (SEQ ID NO: 1),        wherein said second linker is covalently linked to said core via        the thiol group of the non-peptide portion of the linker.

Preferably, the peptide of choice is linked via its N-terminus to saidpeptide portion of said second linker.

In some cases in accordance with the present invention said secondligand is selected from the group consisting of:

-   -   (i) HS—(CH₂)₂—CONH-AAYZ₂;    -   (ii) HS—(CH₂)₂—CONH-FLAAYZ₂ (SEQ ID NO: 1);    -   (iii) HS—(CH₂)₃—CONH-AAYZ₂;    -   (iv) HS—(CH₂)₃—CONH-FLAAYZ₂ (SEQ ID NO: 1);    -   (v) HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-AAYZ₂; and    -   (vi) HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-FLAAYZ₂ (SEQ ID NO: 1),        wherein Z₂ represents said peptide of choice.

In some cases in accordance with the present invention said peptide ofchoice is an epitopic peptide that binds to a class I MajorHistocompatibility Complex (MHC) molecule or is capable of beingprocessed so as to bind to a class I MHC molecule.

In some cases in accordance with the present invention said peptide ofchoice consists of a sequence of 8 to 40 amino acid residues. Inparticular, the peptide of choice may consists of a sequence of 8 to 12amino acid residues.

In some cases in accordance with the present invention the peptide ofchoice is an epitopic peptide that is capable of being presented by aclass I MHC molecule so as to stimulate a Cytotoxic T Lymphocyte (CTL)response.

In some cases in accordance with the present invention the peptide ofchoice forms at least a portion of or is derived from aTumour-Associated Antigen (TAA) or a viral-, bacterial-, orparasite-associated antigen. In particular, the TAA may be a lung cancerantigen. The lung cancer may in some cases be selected from: small-celllung carcinoma and any non-small-cell lung carcinoma, optionally whereinsaid non-small-cell lung carcinoma comprises an adenocarcinoma. Thepathogen-associated antigen may in some cases be a viral, bacterial orparasite antigen.

In some cases in accordance with the present invention, the carbohydratemoiety of said first ligand comprises a monosaccharide and/or adisaccharide. In particular, said carbohydrate moiety may compriseglucose, mannose, fucose and/or N-acetylglucosamine.

In some cases in accordance with the present invention, said pluralityof ligands comprises one or more ligands selected from the groupconsisting of: glucose, N-acetylglucosamine and glutathione, in additionto the one or more ligands comprising said peptide of choice.

In some cases in accordance with the present invention, said pluralityof ligands comprises:

-   -   (a) glucose;    -   (b) N-acetylglucosamine;    -   (c) glutathione;    -   (d) glucose and N-acetylglucosamine;    -   (e) glucose and glutathione;    -   (f) N-acetylglucosamine and glutathione; or    -   (g) glucose, N-acetylglucosamine and glutathione,        in addition to said ligand comprising said peptide of choice.

In some cases in accordance with the present invention said first linkercomprises C2-C15 alkyl and/or C2-C15 glycol.

In some cases in accordance with the present invention said first ligandcomprises 2′-thioethyl-β-D-glucopyranoside or2′-thioethyl-α-D-glucopyranoside covalently attached to the core via thethiol sulphur atom.

In some cases in accordance with the present invention, the nanoparticlecomprises at least 10, at least 20, at least 30, at least 40 or at least50 carbohydrate-containing ligands and/or glutathione ligands.

In some cases in accordance with the present invention the nanoparticlecomprises at least 1, at least 2, at least 3, at least 4 or at least 5peptide-containing ligands.

In some cases in accordance with the present invention, the molar ratioof carbohydrate-containing ligands and/or glutathione ligands topeptide-containing ligands is in the range 5:1 to 100:1, such as in therange 10:1 to 30:1.

In some cases in accordance with the present invention the diameter ofthe core of the nanoparticle is in the range 1 nm to 5 nm.

In some cases in accordance with the present invention the diameter ofthe nanoparticle including its ligands is in the range 5 nm to 20 nm,optionally 5 nm to 15 nm or 8 nm to 10 nm.

In some cases in accordance with the present invention the corecomprises a metal selected from the group consisting of: Au, Ag, Cu, Pt,Pd, Fe, Co, Gd and Zn, or any combination thereof.

In some cases in accordance with the present invention the core ismagnetic.

In some cases in accordance with the present invention the corecomprises a semiconductor. In particular, the semiconductor may in somecases be selected from the group consisting of: cadmium selenide,cadmium sulphide, cadmium tellurium and zinc sulphide.

In some cases in accordance with the present invention the core iscapable of acting as a quantum dot.

In some cases in accordance with the present invention the nanoparticlecomprises at least two peptide of choice-containing ligands, and whereinthe peptide of choice of each of the at least two peptide ofchoice-containing ligands differ.

In some cases in accordance with the present invention the peptides ofchoice of said at least two peptide of choice-containing ligands eachform at least a portion of or are each derived from one or moreantigens, such as TAAs. In some cases in accordance with the presentinvention the peptides of choice of said at least two peptide ofchoice-containing ligands each form at least a portion of or are eachderived from a different lung cancer TAA.

In a further aspect, the present invention provides a compositioncomprising a plurality of nanoparticles in accordance with the firstaspect of the invention. The composition may further comprise at leastone pharmaceutically acceptable carrier, salt and/or diluent.

In some cases in accordance with the present invention the compositioncomprises a first species of nanoparticle having a first peptide ofchoice-containing ligand and a second species of nanoparticle having asecond peptide of choice-containing ligand, wherein the peptides ofchoice of said first and second species differ. The peptides of choiceof each of said first and second species of nanoparticle may in somecases form at least a portion of or each be derived from one or moreantigens, such as TAAs.

In some cases in accordance with the present invention the compositionof the invention may comprise a pool of at least 3, at least 4, at least5 or at least 10 different species of nanoparticle, each species havinga different peptide of choice.

The composition may further comprise at least one adjuvant, whichadjuvant may optionally be covalently attached to the core of at leastone nanoparticle. Alternatively, the composition of the invention may besubstantially free of adjuvant or the only adjuvant effect may beprovided by the nanoparticles.

In a further aspect the present invention provides a vaccine comprisinga composition of the invention. The vaccine may be for therapeutic orprophylactic treatment of a cancer, including lung cancer.

In a further aspect the present invention provides a nanoparticle,composition or vaccine of the invention, for use in medicine.

The nanoparticle, composition or vaccine may be for use in a method oftreatment of a cancer or a pathogen infection in a mammalian subject.

In a further aspect, the present invention provides use of ananoparticle, composition or vaccine of the invention in the preparationof a medicament for the treatment of a cancer or a pathogen infection ina mammalian subject.

The nanoparticle, composition or vaccine of the invention may be foradministration via lymphatic uptake.

In a further aspect, the present invention provides a method ofprophylactic or therapeutic treatment of a cancer or a pathogeninfection, comprising administering a prophylactically ortherapeutically sufficient amount of a nanoparticle, composition orvaccine to a mammalian subject in need thereof. In some cases inaccordance with said method aspect of the present invention, saidnanoparticle, composition or vaccine is administered via a routeselected from the group consisting of:

-   -   injection into an organ or tissue of a mammalian subject at, or        in the vicinity of, a site of lymphatic uptake;    -   nasal delivery via a spray or gel;    -   buccal delivery via a spray or gel or orally dissolvable film;    -   oral delivery via a dissolvable film;    -   transdermal delivery via a patch incorporating the nanoparticle;        and    -   inhalation delivery of a composition comprising the        nanoparticle.

In a further aspect, the present invention provides an in vitro or invivo method for generating a Cytotoxic T Lymphocyte (CTL) response,comprising:

-   -   (i) contacting at least one antigen presenting cell (APC) with        at least one nanoparticle of the invention, such that said        peptide of choice is presented on a class I MHC molecule of said        APC; and    -   (ii) contacting said at least one APC of (i) with at least one        CTL cell, such that said CTL cell is activated by said APC to        generate a CTL response that is specific for said peptide of        choice, wherein said peptide of choice is an epitopic peptide.

In some cases in accordance with the present invention the APC iscultured in the presence of said nanoparticle, and, simultaneously orsequentially, co-cultured with said CTL cell. Optionally, the APC may besubjected to a washing step after being contacted with the nanoparticlebefore being co-cultured with said CTL cell. In some cases, the methodmay further comprise administering the CTL cell to a mammalian subject.

In some cases in accordance with the present invention the nanoparticlemay be delivered by a route of administration selected from the groupconsisting of:

-   -   injection into an organ or tissue of a mammalian subject at, or        in the vicinity of, a site of lymphatic uptake;    -   nasal delivery via a spray or gel;    -   buccal delivery via a spray or gel or orally dissolvable film;    -   oral delivery via a dissolvable film    -   transdermal delivery via a patch incorporating the nanoparticle;        and    -   inhalation delivery of a composition comprising the        nanoparticle.

In some cases, said at least one nanoparticle comprises a pool ofnanoparticles having different peptides of choice.

In a further aspect, the present invention provides a method ofproducing a nanoparticle according of the invention, comprising:

-   -   derivatising the carbohydrate with the first linker;    -   derivatising the peptide of choice with the second linker; and    -   reacting the first linker-derivatised carbohydrate and the        second linker-derivatised peptide of choice with reactants for        producing the core of the nanoparticle so that during        self-assembly of the nanoparticle, the nanoparticle core        attaches the carbohydrate and the peptide of choice via their        respective linkers. In some cases, the reaction mixture        comprises the derivatised carbohydrate, the derivatised peptide        of choice, a salt of the metal and/or semiconductor atoms and a        reducing agent to produce the nanoparticle. Additionally or        alternatively, said carbohydrate may be replaced by, or        supplemented by, glutathione.

In certain cases in accordance with this and other aspects of theinvention the reaction product, including the nanoparticle, is subjectedto a purification step to remove unreacted free peptide, optionallywherein the purification step comprises running the reaction productthrough a G-50 Sephadex™ column.

In certain cases in accordance with this and other aspects of theinvention the method may further comprise the step of formulating thenanoparticle into a composition with at least one pharmaceuticallyacceptable carrier, salt or diluent.

In certain cases in accordance with this and other aspects of theinvention the method is for attaching said peptide of choice via saidsecond linker to said nanoparticle such that the nanoparticle with thepeptide of choice attached is capable of being internalised by a cell.

In certain cases in accordance with this and other aspects of theinvention the method is for attaching said peptide of choice via saidsecond linker to said nanoparticle such that the nanoparticle with thepeptide of choice attached is capable of being internalised by anantigen presenting cell (APC).

In a further aspect, the present invention provides a method forintracellular delivery of a peptide of choice, comprising contacting acell with a nanoparticle or composition of the invention.

In a further aspect, the present invention provides a use of ananoparticle or composition of the invention for the intracellulardelivery of the peptide of choice.

The present invention includes the combination of the aspects andpreferred features described except where such a combination is clearlyimpermissible or is stated to be expressly avoided. These and furtheraspects and embodiments of the invention are described in further detailbelow and with reference to the accompanying examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this application with color drawings will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 shows SIINFEKL (SEQ ID NO: 2) presentation from GNP: LKb cellswere seeded into 24-well plates and allowed to adhere overnight. Next,GNPs were pulsed to the equivalent of 1 ug/mL peptide (Green), 0.1 ug/mL(Blue), or 0.01 ug/mL (Red). After two hours, cells were washed, andsubjected to (A-D) flow cytometric labelling with 25.D1.16 (Angel)antibody, or (E) combined with B3Z CTL for overnight co-culture. Thenext day, cells were lysed and Beta-galactosidase activity was measured.

FIG. 2: GNP presentation compared to free peptide presentation: (A-B)GNPs 8 and 9 were separated by Sephadex column into 15 fractions, whichwere then analyzed by UV absorption for protein levels. The red line(indicated by circles) in FIG. 2A represents free peptide alone. (C-H)LKb cells were pulsed for 2 hrs with noted GNPs or corresponding freepeptide at the noted concentration (1 ug/mL peptide (Green), 0.1 ug/mL(Blue), or 0.01 ug/mL (Red)). Readouts are by (C-F) flow cytometry or(G-H) B3Z assay. (I-N) LKb were pulsed with the noted free peptide for 2hrs on ice (1-K) or at 37° C. (L-N). Next, cells were analyzed by flowcytometry for presentation of SIINFEKL (SEQ ID NO: 2). Red portions ofthe histogram represent positive staining as compared to unpulsed cells.

FIG. 3: GNP presentation from preparations lacking free peptide: (A-B)New preparations of GNPs 8 and 9 were separated by Sephadex G-50 columninto 15 fractions, which were then analyzed by UV absorption for proteinlevels. Arrows indicate which fractions were pooled for further use.(C-E,F) LKb cells were pulsed for 2 hrs with noted previous preparationsof GNPs (old GNP) or newer preparations from (A and B) (new GNP) at thenoted concentrations (1 ug/mL peptide (Green), or 0.1 ug/mL (Red)).Readouts are by (C-E) flow cytometry or (F) B3Z assay.

DETAILED DESCRIPTION OF THE INVENTION

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, “nanoparticle” refers to a particle having a nanomericscale, and is not intended to convey any specific shape limitation. Inparticular, “nanoparticle” encompasses nanospheres, nanotubes,nanoboxes, nanoclusters, nanorods and the like. In certain embodimentsthe nanoparticles and/or nanoparticle cores contemplated herein have agenerally polyhedral or spherical geometry.

Nanoparticles comprising a plurality of carbohydrate-containing ligandshave been described in, for example, WO 2002/032404, WO 2004/108165, WO2005/116226, WO 2006/037979, WO 2007/015105, WO 2007/122388, WO2005/091704 (the entire contents of each of which is expresslyincorporated herein by reference) and such nanoparticles may find use inaccordance with the present invention. Moreover, gold-coatednanoparticles comprising a magnetic core of iron oxide ferrites (havingthe formula XFe₂O₄, where X═Fe, Mn or Co) functionalised with organiccompounds (e.g. via a thiol-gold bond) are described in EP2305310 (theentire contents of which is expressly incorporated herein by reference)and are specifically contemplated for use as nanoparticles/nanoparticlecores in accordance with the present invention.

As used herein, “corona” refers to a layer or coating, which maypartially or completely cover the exposed surface of the nanoparticlecore. The corona includes a plurality of ligands which include at leastone carbohydrate moiety, one surfactant moiety and/or one glutathionemoiety. Thus, the corona may be considered to be an organic layer thatsurrounds or partially surrounds the metallic core. In certainembodiments the corona provides and/or participates in passivating thecore of the nanoparticle. Thus, in certain cases the corona may includea sufficiently complete coating layer substantially to stabilise themetal-containing core. However, it is specifically contemplated hereinthat certain nanoparticles having cores, e.g., that include a metaloxide-containing inner core coated with a noble metal may include acorona that only partially coats the core surface. In certain cases thecorona facilitates solubility, such as water solubility, of thenanoparticles of the present invention.

Nanoparticles

Nanoparticles are small particles, e.g. clusters of metal orsemiconductor atoms, that can be used as a substrate for immobilisingligands.

Preferably, the nanoparticles have cores having mean diameters between0.5 and 50 nm, more preferably between 0.5 and 10 nm, more preferablybetween 0.5 and 5 nm, more preferably between 0.5 and 3 nm and stillmore preferably between 0.5 and 2.5 nm. When the ligands are consideredin addition to the cores, preferably the overall mean diameter of theparticles is between 5.0 and 100 nm, more preferably between 5 and 50 nmand most preferably between 5 and 10 nm. The mean diameter can bemeasured using techniques well known in the art such as transmissionelectron microscopy.

The core material can be a metal or semiconductor and may be formed ofmore than one type of atom. Preferably, the core material is a metalselected from Au, Fe or Cu. Nanoparticle cores may also be formed fromalloys including Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd andAu/Fe/Cu/Gd, and may be used in the present invention. Preferred corematerials are Au and Fe, with the most preferred material being Au. Thecores of the nanoparticles preferably comprise between about 100 and 500atoms (e.g. gold atoms) to provide core diameters in the nanometerrange. Other particularly useful core materials are doped with one ormore atoms that are NMR active, allowing the nanoparticles to bedetected using NMR, both in vitro and in vivo. Examples of NMR activeatoms include Mn⁺², Gd⁺³, Eu⁺², Cu⁺², V⁺², Co⁺², Ni⁺², Fe⁺², Fe⁺³ andlanthanides⁺³, or the quantum dots described elsewhere in thisapplication.

Nanoparticle cores comprising semiconductor atoms can be detected asnanometer scale semiconductor crystals are capable of acting as quantumdots, that is they can absorb light thereby exciting electrons in thematerials to higher energy levels, subsequently releasing photons oflight at frequencies characteristic of the material. An example of asemiconductor core material is cadmium selenide, cadmium sulphide,cadmium tellurium. Also included are the zinc compounds such as zincsulphide.

In some embodiments, the core of the nanoparticles may be magnetic andcomprise magnetic metal atoms, optionally in combination with passivemetal atoms. By way of example, the passive metal may be gold, platinum,silver or copper, and the magnetic metal may be iron or gadolinium. Inpreferred embodiments, the passive metal is gold and the magnetic metalis iron. In this case, conveniently the ratio of passive metal atoms tomagnetic metal atoms in the core is between about 5:0.1 and about 2:5.More preferably, the ratio is between about 5:0.1 and about 5:1. As usedherein, the term “passive metals” refers to metals which do not showmagnetic properties and are chemically stable to oxidation. The passivemetals may be diamagnetic or superparamagnetic. Preferably, suchnanoparticles are superparamagnetic.

Examples of nanoparticles which have cores comprising a paramagneticmetal, include those comprising Mn⁺², Gd⁺³, Eu⁺², Cu⁺², V⁺², Co⁺², Ni⁺²,Fe⁺², Fe⁺³ and lanthanides⁺³.

Other magnetic nanoparticles may be formed from materials such as MnFe(spinel ferrite) or CoFe (cobalt ferrite) can be formed intonanoparticles (magnetic fluid, with or without the addition of a furthercore material as defined above. Examples of the self-assembly attachmentchemistry for producing such nanoparticles is given in Biotechnol.Prog., 19:1095-100 (2003), J. Am. Chem. Soc. 125:9828-33 (2003), J.Colloid Interface Sci. 255:293-8 (2002).

In some embodiments, the nanoparticle or its ligand comprises adetectable label. The label may be an element of the core of thenanoparticle or the ligand. The label may be detectable because of anintrinsic property of that element of the nanoparticle or by beinglinked, conjugated or associated with a further moiety that isdetectable. Preferred examples of labels include a label which is afluorescent group, a radionuclide, a magnetic label or a dye.Fluorescent groups include fluorescein, rhodamine or tetramethylrhodamine, Texas-Red, Cy3, Cy5, etc., and may be detected by excitationof the fluorescent label and detection of the emitted light using Ramanscattering spectroscopy (Y. C. Cao, R. Jin, C. A. Mirkin, Science 2002,297: 1536-1539).

In some embodiments, the nanoparticles may comprise a radionuclide foruse in detecting the nanoparticle using the radioactivity emitted by theradionuclide, e.g. by using PET, SPECT, or for therapy, i.e. for killingtarget cells. Examples of radionuclides commonly used in the art thatcould be readily adapted for use in the present invention include^(99m)Tc, which exists in a variety of oxidation states although themost stable is TcO⁴⁻; ³²P or ³³P; ⁵⁷Co; ⁵⁹Fe; ⁶⁷Cu which is often usedas Cu²⁺ salts; ⁶⁷Ga which is commonly used a Ga³⁺ salt, e.g. galliumcitrate; ⁶⁸Ge; ⁸²Sr; ⁹⁹Mo; ¹⁰³Pd; ¹¹¹In which is generally used as In³⁺salts; ¹²⁵I or ¹³¹I which is generally used as sodium iodide; ¹³⁷Cs;¹⁵³Gd; ¹⁵³Sm; ¹⁵⁸Au; ¹⁸⁶Re; ²⁰¹Tl generally used as a Tl⁺ salt such asthallium chloride; ³⁹Y³⁺; ⁷¹Lu³⁺; and ²⁴Cr²⁺. The general use ofradionuclides as labels and tracers is well known in the art and couldreadily be adapted by the skilled person for use in the aspects of thepresent invention. The radionuclides may be employed most easily bydoping the cores of the nanoparticles or including them as labelspresent as part of ligands immobilised on the nanoparticles.

Additionally or alternatively, the nanoparticles of the presentinvention, or the results of their interactions with other species, canbe detected using a number of techniques well known in the art using alabel associated with the nanoparticle as indicated above or byemploying a property of them. These methods of detecting nanoparticlescan range from detecting the aggregation that results when thenanoparticles bind to another species, e.g. by simple visual inspectionor by using light scattering (transmittance of a solution containing thenanoparticles), to using sophisticated techniques such as transmissionelectron microscopy (TEM) or atomic force microscopy (AFM) to visualisethe nanoparticles. A further method of detecting metal particles is toemploy plasmon resonance that is the excitation of electrons at thesurface of a metal, usually caused by optical radiation. The phenomenonof surface plasmon resonance (SPR) exists at the interface of a metal(such as Ag or Au) and a dielectric material such as air or water. Aschanges in SPR occur as analytes bind to the ligand immobilised on thesurface of a nanoparticle changing the refractive index of theinterface. A further advantage of SPR is that it can be used to monitorreal time interactions. As mentioned above, if the nanoparticles includeor are doped with atoms which are NMR active, then this technique can beused to detect the particles, both in vitro or in vivo, using techniqueswell known in the art. Nanoparticles can also be detected using a systembased on quantitative signal amplification using thenanoparticle-promoted reduction of silver (I). Fluorescence spectroscopycan be used if the nanoparticles include ligands as fluorescent probes.Also, isotopic labelling of the carbohydrate can be used to facilitatetheir detection.

Peptide of Choice

The nanoparticle of the present invention comprises a peptide-containingligand (“said second ligand”) that is covalently linked to the core ofthe nanoparticle via a linker (“said second linker”). The second ligandcomprises a peptide that may be intended for intracellular delivery.This “peptide of choice” may be any suitable peptide. In particular, thepeptide may be a fragment of a larger polypeptide that is able to exertan effect when delivered to an intracellular site. In certain cases, thepeptide of choice may be a peptide-based drug. In certain cases, thepeptide of choice may comprise an epitope (an “epitopic peptide”) thatgives rise to an immune response when delivered to, for example, anintracellular compartment of an APC. In certain preferred cases, thepeptide of choice may be a peptide that binds to a class I MajorHistocompatibility Complex (MHC) molecule or is capable of beingprocessed so as to bind to a class I MHC molecule. In some cases inaccordance with the present invention, the peptide of choice may consistof a sequence of 8 to 40 amino acid residues, such as a sequence of 8 to12 amino acid residues. In certain cases, the peptide of choice may becapable of being presented by a class I MHC molecule so as to stimulatea Cytotoxic T Lymphocyte (CTL) response. Such epitopic peptides may format least a portion of, or be derived from, a Tumour-Associated Antigen(TAA) or a pathogen-associated antigen (e.g. a viral, bacterial orparasite antigen). In some cases, the peptide of choice may be a peptidedrug, such as a peptide that targets an intracellular target. Oneexample of such a intracellular targeting peptide is the GIPC-PDZ domaininhibitor described in Muders et al., 2009, Clin Cancer Res, Vol.15(12), pp. 4095-4103 (the entire contents of which is herebyincorporated herein by reference).

Administration and Treatment

The nanoparticles and compositions of the invention may be administeredto patients by any number of different routes, including enteral orparenteral routes. Parenteral administration includes administration bythe following routes: intravenous, cutaneous or subcutaneous, nasal,intramuscular, intraocular, transepithelial, intraperitoneal and topical(including dermal, ocular, rectal, nasal, inhalation and aerosol), andrectal systemic routes.

Administration be performed e.g. by injection, or ballistically using adelivery gun to accelerate their transdermal passage through the outerlayer of the epidermis. The nanoparticles can then be taken up, e.g. bydendritic cells, which mature as they migrate through the lymphaticsystem, resulting in modulation of the immune response and vaccinationagainst the epitopic peptide and/or the antigen from which the epitopicpeptide was derived or of which it forms a part. The nanoparticles mayalso be delivered in aerosols. This is made possible by the small sizeof the nanoparticles.

The exceptionally small size of the nanoparticles of the presentinvention is a great advantage for delivery to cells and tissues, asthey can be taken up by cells even when linked to targeting ortherapeutic molecules. Thus, the nanoparticles may be internalised bycells such as APCs, the peptides processed and released, e.g. forpresentation via class I MHC or for interaction with intracellularcomponents.

The nanoparticles of the invention may be formulated as pharmaceuticalcompositions that may be in the forms of solid or liquid compositions.Such compositions will generally comprise a carrier of some sort, forexample a solid carrier such as gelatine or an adjuvant or an inertdiluent, or a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, or glycols such as ethylene glycol, propylene glycol orpolyethylene glycol may be included. Such compositions and preparationsgenerally contain at least 0.1 wt % of the compound.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,solutions of the compounds or a derivative thereof, e.g. inphysiological saline, a dispersion prepared with glycerol, liquidpolyethylene glycol or oils.

In addition to one or more of the compounds, optionally in combinationwith other active ingredient, the compositions can comprise one or moreof a pharmaceutically acceptable excipient, carrier, buffer, stabiliser,isotonicising agent, preservative or anti-oxidant or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material maydepend on the route of administration, e.g. orally or parenterally.

Liquid pharmaceutical compositions are typically formulated to have a pHbetween about 3.0 and 9.0, more preferably between about 4.5 and 8.5 andstill more preferably between about 5.0 and 8.0. The pH of a compositioncan be maintained by the use of a buffer such as acetate, citrate,phosphate, succinate, Tris or histidine, typically employed in the rangefrom about 1 mM to 50 mM. The pH of compositions can otherwise beadjusted by using physiologically acceptable acids or bases.

Preservatives are generally included in pharmaceutical compositions toretard microbial growth, extending the shelf life of the compositionsand allowing multiple use packaging. Examples of preservatives includephenol, meta-cresol, benzyl alcohol, para-hydroxybenzoic acid and itsesters, methyl paraben, propyl paraben, benzalconium chloride andbenzethonium chloride. Preservatives are typically employed in the rangeof about 0.1 to 1.0% (w/v).

Preferably, the pharmaceutically compositions are given to an individualin a prophylactically effective amount or a therapeutically effectiveamount (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual.Typically, this will be to cause a therapeutically useful activityproviding benefit to the individual. The actual amount of the compoundsadministered, and rate and time-course of administration, will depend onthe nature and severity of the condition being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Handbook of Pharmaceutical Additives,2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse InformationResources, Inc., Endicott, N.Y., USA); Remington's PharmaceuticalSciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; andHandbook of Pharmaceutical Excipients, 2nd edition, 1994. By way ofexample, and the compositions are preferably administered to patients indosages of between about 0.01 and 100 mg of active compound per kg ofbody weight, and more preferably between about 0.5 and 10 mg/kg of bodyweight.

It will be understood that where treatment of tumours is concerned,treatment includes any measure taken by the physician to alleviate theeffect of the tumour on a patient. Thus, although complete remission ofthe tumour is a desirable goal, effective treatment will also includeany measures capable of achieving partial remission of the tumour aswell as a slowing down in the rate of growth of a tumour includingmetastases. Such measures can be effective in prolonging and/orenhancing the quality of life and relieving the symptoms of the disease.

Immunotherapy

The compositions of the invention, such as the vaccines as defined inthe claims, may in some cases be used for the prophylaxis and treatmentof diseases such as cancer, and more particularly for immunotherapy.

In the present invention, the term “vaccination” means an activeimmunization, that is an induction of a specific immune response due toadministration, e.g. via the subcutaneous, intradermal, intramuscular,oral or nasal routes, of small amounts of an antigen which is recognizedby the vaccinated individual as foreign and is therefore immunogenic ina suitable formulation. The antigen is thus used as a “trigger” for theimmune system in order to build up a specific immune response againstthe antigen.

In accordance with the present invention, vaccination may be therapeuticor prophylactic. By way of example, it might be possible to achieve aprophylactic protection against the breakout of a cancer disease byvaccination of individuals who do not suffer from cancer. Examples ofindividuals for whom such a prophylactic vaccination might be appliedare individuals who have an increased risk of developing a cancerdisease, although this application is not limited to such individuals.Patients being at risk of cancer can already have developed tumours,either as primary tumours or metastases, or show predisposition forcancer.

For the active immunization of cancer patients according to theinvention, the nanoparticles are typically formulated as vaccines.Preferably, such pharmaceutical preparations contain a pharmaceuticallyacceptable carrier which, by way of example, may further compriseauxiliary substances, buffers, salts and/or preserving agents. Thepharmaceutical preparations may, e.g., be used for the prophylaxis andtherapy of cancer-associated conditions, such as metastasis formation,in cancer patients. In doing so, antigen-presenting cells arespecifically modulated in vivo or also ex vivo so as to generate theimmune response against the TAAs.

For the active immunization with the specific antigens or the antigencombination usually a vaccine formulation is used which contains theimmunogen—be it a natural TAA or its epitope, mimic or neoepitopemimic,—mostly at low concentrations, e.g. in an immunogenic amountranging from 0.01 μg to 10 mg, yet the dosage range can be increased upa range of 100 to 500 mg. Depending on the immunogenicity of thevaccination antigen which is, e.g., determined by sequences of a foreignspecies or by derivatization, or also depending on the auxiliarysubstances or adjuvants, respectively, used, the suitable immunogenicdose can be chosen e.g. in the range of from 0.01 μg to 1 mg, preferably100 μg to 500 μg. A depot vaccine which is to be delivered to theorganism over an extended period of time may, however, also contain muchhigher amounts of vaccination antigen, e.g. at least 1 mg to more than100 mg.

The concentration will depend on the amount of liquid or suspendedvaccine administered. A vaccine usually is provided in ready-to-usesyringes or ampoules having a volume ranging from 0.01 to 1 ml,preferably 0.1 to 0.75 ml.

The vaccination antigen of a component of vaccine preferably ispresented in a pharmaceutically acceptable carrier which is suitable forsubcutaneous, intramuscular and also intradermal or transdermaladministration. A further mode of administration functions via themucosal pathway, e.g. vaccination by nasal or peroral administration. Ifsolid substances are employed as auxiliary agent for the vaccineformulation, e.g. an adsorbate, or a suspended mixture, respectively, ofthe vaccine antigen with the auxiliary agent will be administered. Inspecial embodiments, the vaccine is presented as a solution or a liquidvaccine in an aqueous solvent.

Preferably, vaccination units of a tumour vaccine are already providedin a suitable ready-to-use syringe or ampoule. A stable formulation ofthe vaccine may advantageously be put on the market in a ready to useform. Although a content of preserving agents, such as thimerosal orother preserving agents with an improved tolerability, is notnecessarily required, yet it may be provided in the formulation for alonger stability at storage temperatures of from refrigeratingtemperatures up to room temperature. The vaccine according to theinvention may, however, also be provided in frozen or lyophilized formand may be thawed or reconstituted, respectively, upon demand.

In certain cases in accordance with the present invention theimmunogenicity of the vaccine of the invention may be increased by byemploying adjuvants. For this purpose, solid substances or liquidvaccine adjuvants are used, e.g. aluminum hydroxide (Alu-Gel) oraluminum phosphate, growth factors, lymphokines, cytokines, such asIL-2, IL-12, GM-CSF, gamma interferon, or complement factors, such asC3d, further liposome preparations, or also formulations with additionalantigens against which the immune system has already generated a strongimmune response, such as tetanus toxoid, bacterial toxins, such asPseudomonas exotoxins, and derivatives of lipid A andlipopolysaccharide.

In certain cases, no additional adjuvant is employed, in particular, theexamples described herein show that efficient generation of a CTLresponse is achieved using nanoparticles of the invention without anyadded adjuvant (c.f. free peptides which are generally administered withone or more adjuvants). This is highly beneficial in certaincircumstances as a number of adjuvants are considered to be toxic orotherwise unsuitable for human use.

The following is presented by way of example and is not to be construedas a limitation to the scope of the claims.

EXAMPLES Example 1 Synthesis and Characterisation of Nanoparticles

Test ligands and their identification numbers are given below (Molecularwt);

(SEQ ID NO: 2) SIINFEKL (963) (SEQ ID NO: 2) SIINFEKL-N—(CH₂)₂—SH (1021)(SEQ ID NO: 3) FLSIINFEKL-N—(CH₂)₂—SH (1280) (SEQ ID NO: 4)FLAAYSIINFEKL-N—(CH₂)₂—SH (1587) (SEQ ID NO: 5)AAYSIINFEKL-N—(CH₂)₂—SH (1325) (SEQ ID NO: 2)HS(CH₂)₂—CONH-SIINFEKL (1051)   (SEQ ID NO: 3)HS(CH₂)₂—CONH-FLSIINFEKL (1309) (SEQ ID NO: 4)HS(CH₂)₂—CONH-FLAAYSIINFEKL (1616) (SEQ ID NO: 5)HS(CH₂)₂—CONH-AAYSIINFEKL (1356) (SEQ ID NO: 2)HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-SIINFEKL (1471) (SEQ ID NO: 3)HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-FLSIINFEKL (1732) (SEQ ID NO: 4)HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-FLAAYSIINFEKL (2034) (SEQ ID NO: 5)HS—(CH₂)₁₀—(CH₂OCH₂)₇—CONH-AAYSIINFEKL (1774)

Test NPs were synthesized using 10 μmole Gold Chloride (Aldrich 484385),30 μmole glucose with a thio ethyl linker (GlcC2) and 1.5 μmole ofpeptide ligand (variable 1.5-3 mg).

The following method was used; 1.5 μmole peptide was dissolved in 2 mlmethanol, followed by the addition of 30 μmole GlcC2 in 200 μl methanol,and 116 μl of aqueous gold chloride containing 10 μmole Au. The samplewas vortexed for 30 sec, shaken for approximately 5 min and then underrapid vortexing for a total of 30 sec 200 μl of 1M NaBH₄ was added,tubes were sealed and then gently shaken for 1.5 h.

Samples were bench spun the supernatant removed and the dark pelletdissolved in 2 ml water, and then transferred to a 10 kDa vivaspin for atotal of 4 times 2 ml water washes each of 8 min at 5 Krpm.

Nanoparticles (NPs) were removed from the vivaspins and made up to 500μl with water, they were then subjected to 15 Krpm bench spin to removeany large aggregates.

Gold content post spin/production by in house assay is shown below 100%yield would be 1.97 mg;

NP Total mg Au 2 1.14 3 0.03 4 0.00 5 0.05 6 1.34 7 1.06 9 1.59 10  1.6911  1.37 13  1.49 Glc 1.02

NPs 3, 4 and 5 showed large near complete aggregation, addition of DMSOto these aggregates failed to solubilise these particle.

The remaining NP samples were respun to remove any further aggregates,and aliquots taken to allow for later analyses, specifically gold andpeptide content. One aliquot was made up to 500 μl with water, dated andlabelled, these were subsequently used for HLA presentation testing.

The final analysis of these 500 μl samples for HLA presentation was asfollows;

Total μmole nmole NP Au nmole peptide* peptide** 2 1.46 166 106 3 — — —4 — — — 5 — — — 6 3.09 348 263 7 3.36 287 219 9 4.77 508 286 10  4.42256 372 11  3.26 235 830 13  4.19 386 1083  Glc 3.11 nd nd *BSA Std**Peptide Std

Ligand ratios used were such that 2 peptides should theoretically beattached to each NP of approximately 100 Au atoms, the data abovesuggests approximately 6-8 peptides/100 Au atoms. This could simple bean artefact of BCA method (and BSA standard) used for peptidemeasurement of NP bound peptides or perhaps the peptide ligands attach.

Analysis of peptide content is both crucial and in this case complex,the BCA method was used. Unfortunately gold NPs exhibit large uv/visabsorbance, so in addition to running the test samples aliquots of NPswere also measured in water and blanked against water to determine theirabsorbance at 565 nm the wavelength used in the BCA assay, thisabsorbance amounted to approximately 20% of the test sample BCA assayvalue and was individually corrected for. This correction however,assumes that a peptide NP subjected to BCA analysis will still maintainthe same absorbance seen for the free NP on top of the BCA specificcomponent; this is consistent with the high extinction coefficients seenwith just pure gold NPs.

In the table above * refers to quantitation related to a BSA standardinitially on a weight basis then converted to moles of peptide, in the** column the individual peptide ligands were used as standards.

Initial analyses using Sephadex G-50 with PBS elution to try to resolvefree form NP bound peptide was performed primarily with NP6, suggestthat little/no free ligand is contaminating the NP preparations. Iodinewas used to release all NP bound ligands the iodine began to appear inthe fractions 16+ for FIG. 1.

Larger fraction sizes and equivalent amounts of NP pre and post iodinetreatment were then used, the iodine released peptide elutes with a peakin fraction 10, this material appears smaller than the standard peptidepossibly because the latter is oxidized/dimeric. Individual correctionswere also applied for non peptidic absorbances from NP6, near equivalentareas under the curve were obtained for NP6 corrected and NP6+iodine.

Production of NP8 and 12

NP8 and 12 were successfully produced by the methods above,quantitations given below represent the 500 μl sample subsequently used,which in turn is (60% of the total preparation);

nmole NP μmole Au peptide* 8 5.28 175 12 3.21 65 *Determined byCoomassie method

Repeat Production of NPs 2-5

NP's 2-5 were produced by a variation using 75% methanol not 95% in thesynthesis stage. NP2 produced a ‘normal’ NP as previously, NPs 3, 4 and5 as before formed aggregates, these aggregates where not soluble inwater, 10% acetic acid, PBS, DMSO or DMF, however 300 mM NaOH did resultin total NP solubilisation.

Additional synthesis was carried out using Sephadex G-50 to remove anyfree peptide. These NPs were made as above but with the followingchanges:

The free peptides were not fully soluble in MeOH so in addition to the 2ml MeOH 100 μl water was added plus for P8 70 μl of 1MNaOH and for P9only 20 μl NaOH that was required to effect full peptide solubilisation.NaOH is compatible with NP synthesis, the water and alkali reduced thefinal MeOH % post borohydride reduction to 82 and 83.5% for NP8 and NP9respectively.

An extra wash step was included after the initial spin down postreduction but pre-vivaspin, this was performed with 2 ml MeOH and 100 μlof 1MNaCl.

Post vivaspin half of each preparation was subjected to a Sephadex G-50column eluted with PBS, fractions collected and aliquots measured forprotein by BCA, tight pools of the main proteinaceous peaks were pooledand then resubjected to vivaspin with water washes to effect solventchange.

Only half of the NP preparation was passed down G-50, the resultantprofiles and pools were found to be essentially clear of free peptide.The NP brown colouration could be visually seen up to fraction 12, in aseparate run 50 ul of the stock preparation was rerun under the sameconditions and the 515 nm absorbance measured (which will detect Au NPsnot free peptides) and gives an indication if the NP is trailing off theG-50 column.

The final pooled material had the following specifications;

NP8 NP9 Volume ml 0.5 0.5 Peptide by BCA (BSA 638 490 std) μg/mlTheoretical peptide 225 118 μg/ml Au mg/ml 1.30 0.81

The theoretical values are determined by assuming 44 ligands/100 Au,random competition between the two ligands at the time of NP formationand the Au yield.

NP Ligand Release with Iodide

An aliquot of NP9 was mixed with a 4-fold volume excess of 1M KI andleft at 4° C. for 4 days, in order to result in complete ligand release.After 4 days, the material was centrifuged and the clear supernatantpassed down G-50 and fractions collected and assayed by BCA. The amountof NP9 post KI assayed was exactly twice that used for the NP9 alone (acorrection of 1.1 was applied for inter-assay absorbance differences),the areas were found to be almost exactly 2:1 suggesting complete ligandremoval. The amount of ligand released was quantitated using 2 assays;Coomassie and BCA in conjunction with 2 standards peptide 9 and BSA, andis tabulated below.

Protein std used Coomassie μg BCA μg BSA 377 611 P9 2210 1062

The data in the table has been corrected up to the total expected yieldfor the whole preparation.

In conclusion, peptide-containing NPs have been synthesized. The peptideNPs are essentially devoid of contaminating free peptides by simple useof Sephadex G-50 gel filtration chromatography. Iodide was successfullyused to release NP bound peptide, and gave quantitative yield data.

Example 2 Evaluation of Presentation Assays

T cell receptors (TCR) are on the surface of T lymphocytes and recognizepeptides in the context of major histocompatibility complex (MHC) (1).Generally, antigen presenting cells (APC) contain machinery to processproteins and load them onto empty MHC. While CD4+ T cells recognize MHCClass II (MHCII), CD8+ T cells respond to MHC Class I (MHCI).Conventionally, MHCII peptides derive from endocytosed components of theextracellular milieu. In contrast, MHCI loads peptides processed from anintracellular source (1, 2).

SIINFEKL (SEQ ID NO: 2), a peptide epitope that is derived fromovalbumin (OVA), is presented in the context of a murine MHCI alleletermed H-2K^(b) (3). If OVA is expressed in a murine cell expressingH-2K^(b), SIINFEKL is presented conventionally. However, if OVA issupplied exogenously, SIINFEKL can be presented by an alternativeprocess known as MHCI cross-presentation (4, 5). In fact,haplotype-matched mouse immunized with OVA generate an immunodominantresponse to SIINFEKL (SEQ ID NO: 2).

Therefore, many reagents have been developed to assay the presentationof this peptide in an effort to further the understanding ofconventional and alternative MHCI presentation. These reagents can beutilized to experiment with the potential chemical linkages of peptidesin nanoparticles. Thus, we can uncover which linkage is most easilyprocessed in a mouse cell line.

Results and Discussion

Flow Cytometry as a Measure of Presentation

In order to analyze epitope linkage to nanoparticles, we first optimizeda readout assay for the presentation of the epitope released from thenanoparticle. For this purpose, SIINFEKL (SEQ ID NO: 2) presentation inLK^(b) cells was evaluated. As mentioned earlier, more than one reagentexists. Of the two methods tested, one is flow cytometry-based, whilethe other is cell-based. The flow cytometry-based method begins withpulsing LK^(b) cells with differing amounts of SIINFEKL (SEQ ID NO: 2)peptide. After allowing enough time for peptide binding to surface K^(b)molecules, cells were washed and incubated with the 25.D1.16 antibodywhich is specific to the SIINFEKL:K^(b) (SEQ ID NO: 2) complex. Next,the cells were secondarily labelled and subjected to flow cytometryanalysis using unpulsed cells as the background reading. It was foundthat this method detected surface complexes when the cells were pulsedwith as little as 5 ng/mL.

T Cell Activation as a Measure of Antigen Presentation

Another form of epitope-specific antigen presentation is the measurementof T cell activation by the MHCI peptide complex. Here, we used B3Z (OVApeptide specific T cell line), which recognizes SIINFEKL (SEQ ID NO: 2)the context of K^(b). As a convenient measure, this T cell line containsβ-galactosidase cloned with the NFAT promoter. Upon peptide recognitionand T cell activation, β-galactosidase is expressed and conversion of adetectible substrate serves as an excellent measure of antigenpresentation to T cells.

To evaluate this method, we performed a similar experiment as above.LK^(b) cells were pulsed with SIINFEKL (SEQ ID NO: 2) peptide, washed,and then co-incubated with B3Z T cell line overnight. The next day,cells were lysed and β-galactosidase was measured using a luminescentsubstrate. As expected, the resolution of this method was similar to theprevious method with the limit of detection at approximately 5 ng/mL.

Therefore, both methods exhibit approximately the same resolution andwere selected for use in the evaluation of nanoparticle-peptideconstructs.

Materials and Methods

Cell Lines

LK^(b) cells are mouse fibroblasts and were the primary line used.Specifically, they are L929 cells stably expressing the murine H-2K^(b)molecule.

Synthetic Peptides

Synthetic SIINFEKL (OVA 257-264) (SEQ ID NO: 2) peptides were purchasedfrom Genscript USA (Piscataway, N.J.). Peptides were resuspended to 5mg/mL in DMSO and pulsed onto cells at the concentration denoted in thefigures.

T Cell Hybridomas

The SIINFEKL:Kb-specific T hybridoma (B3Z) (SEQ ID NO: 2) expresses(β-galactosidase upon recognition of peptide-MHC class I complexes andhas been described previously (3, 6). T cell hybridomas were maintainedin complete RPMI plus 10% FCS and 0.05 mM 2-ME. Activation was measuredusing the luminescent substrate Galactolight Plus (Applied Biosystems,Foster City, Calif.) according to the manufacturer's instructions. Lightintensity was measured using a TopCount NXT plate reader (Perkin Elmer,Waltham, Mass.).

Flow Cytometry

LK^(b) cells were treated with varying amounts of SIINFEKL (SEQ ID NO:2) peptides. After a 2 hr incubation, cells were collected, washed oncewith PBS, and then incubated for 1 hr with 25.D1.16 culture supernatant(monoclonal antibody specific for SIINFEKL (SEQ ID NO: 2) complexed toH-2K^(b)) (7) on ice. Cells were then washed two times in PBS andincubated for 30 min on ice with FITC-labeled goat anti-mouse IgGsecondary antibody (Caltag Laboratories, Burlingham, Calif.). Finally,cells were washed two times with PBS and resuspended in PBS+0.1% BSA forflow cytometry on a Guava EasyCyte Plus Flow Cytometer (Millipore,Billerica, Mass.) and analyzed using the accompanying software.

Antigen Presentation Assays

To assay SIINFEKL (SEQ ID NO: 2) presentation using a flow cytometric orcell-based method, we pulsed LK^(b) cells in a 15 mL conical at 37° C.for 2 hrs. Following this incubation, cells were washed once with PBSand then subjected to detection using flow cytometry or added to B3Zcells at an effector to target ratio of 1:1.

REFERENCES

-   1. Vyas, J. M., A. G. Van der Veen, and H. L. Ploegh. 2008. The    known unknowns of antigen processing and presentation. Nature    reviews 8:607-618.-   2. Hansen, T. H., and M. Bouvier. 2009. MHC class I antigen    presentation: learning from viral evasion strategies. Nature reviews    9:503-513.-   3. Shastri, N., and F. Gonzalez. 1993. Endogenous generation and    presentation of the ovalbumin peptide/K^(b) complex to T cells.    Journal of Immunology 150:2724-2736.-   4. Amigorena, S., and A. Savina. 2010. Intracellular mechanisms of    antigen cross presentation in dendritic cells. Current opinion in    immunology 22:109-117.-   5. Blanchard, N., and N. Shastri. 2010. Cross-presentation of    peptides from intracellular pathogens by MHC class 1 molecules.    Annals of the New York Academy of Sciences 1183:237-250.-   6. Tewari, M. K., G. Sinnathamby, D. Rajagopal, and L. C.    Eisenlohr. 2005. A cytosolic pathway for MHC class II-restricted    antigen processing that is proteasome and TAP dependent. Nature    immunology 6:287-294.-   7. Porgador, A., J. W. Yewdell, Y. Deng, J. R. Bennink, and R. N.    Germain. 1997. Localization, quantitation, and in situ detection of    specific peptide-MHC class I complexes using a monoclonal antibody.    Immunity 6:715-726.

Example 3 Nanoparticle-Peptide Presentation Assays

The test ligands listed below were constructed and attached to goldnanoparticles (GNP) by the above-described linker chemistry.

(SEQ ID NO: 2)  1. SIINFEKL  2. SIINFEKL-N—(CH₂)₂-SH (SEQ ID NO: 3) 3. FLSIINFEKL-N—(CH₂)₂-SH (SEQ ID NO: 4)  4. FLAAYSIINFEKL-N—(CH₂)₂-SH(SEQ ID NO: 5)  5. AAYSIINFEKL-N—(CH₂)₂-SH (SEQ ID NO: 2) 6. HS(CH2)2-CONH-SIINFEKL (SEQ ID NO: 3)  7. HS(CH₂)₂-CONH-FLSIINFEKL(SEQ ID NO: 4)  8. HS(CH₂)₂-CONH-FLAAYSIINFEKL (SEQ ID NO: 5) 9. HS(CH2)2-CONH-AAYSIINFEKL (SEQ ID NO: 2)10. HS—(CH2)10-(CH2OCH2)7-CONH-SIINFEKL (SEQ ID NO: 3)11. HS—(CH2)10-(CH2OCH2)7-CONH-FLSIINFEKL (SEQ ID NO: 4)12. HS—(CH2)10-(CH2OCH2)7-CONH-FLAAYSIINFEKL (SEQ ID NO: 5)13. HS—(CH2)10-(CH2OCH2)7-CONH-AAYSIINFEKL

SIINFEKL (SEQ ID NO: 2), an epitope derived from ovalbumin that ispresented in the context of the murine MHCI molecule H-2 Kb, wasmeasured using two methods. One method utilized a TCR-like antibodytermed 25.D1.16, also referred to as “Angel”, that recognize SIINFEKL(SEQ ID NO: 2)/MHCI complex. In addition, we assessed presentation usingthe B3Z, SIINFEKL (SEQ ID NO: 2) peptide specific CTL hybridoma, whichexpresses β-galactosidase under the NFAT (CTL signaling molecule)promoter, which upon activation express beta-gal measured by a lightemitting substrate.

A. Analysis of GNP

To assess the ability of cells to process and present SIINFEKLassociated with GNP, we used L-Kb murine fibroblasts. As demonstrated inFIG. 1, GNPs 8, 9, 12, and 13 displayed good processing andpresentation. This was found to be the case for both flow cytometric(FIG. 1A-D—processing) and CTL-based methods (FIG. 1E—presentation).Thus, FLAAYSIINFEKL (SEQ ID NO: 4) and AAYSIINFEKL (SEQ ID NO: 5)exhibited superior in vitro SIINFEKL (SEQ ID NO: 2) processing (theunderlined portion showing the peptide portion of the linker. Also, asillustrated in FIG. 1E, HS—(CH2)10-(CH2OCH2)7-CONH chemistry was foundto be better processed than HS(CH2)2-CONH. However, HS(CH2)2-CONH wasstill found to be processed very efficiently. Additionally, we note adose-dependent reduction of presentation with no detection at 0.01μg/mL.

B. Analysis of Free Peptide

It was important to consider the possible effects of any free peptidepresent in the nanoparticle samples (FIGS. 2A-B). Therefore, weevaluated how efficiently corresponding free peptide from eachpreparation is presented. Similar to previous experiments, LKb cellswere pulsed for 2 hrs with three concentrations of GNP or free peptide.Presentation was assessed by flow cytometry (FIGS. 2C-F) or B3Z assay(FIGS. 2G-H). From these results, we deduced that free peptide ispresented well. Lastly, we sought to test whether processing isnecessary for presentation of these free peptides. A test requires thepulsing of peptides on ice compared to 37° C. At 37° C., cells can takeup peptide and process it within the endosome, however, on ice, peptidecan only be loaded on the surface without processing. Indeed, weobserved that free peptides with linkers generally need processing whilethe peptide without the linker can be presented (FIGS. 2I-N) without anyprocessing.

C. Analysis of Preparations Lacking Free Peptide

In view of the results demonstrating the possible effect ofcontaminating free peptides, purified preparations were made.Purification was carried out on the GNPs (8 and 9) using a Sephadex G-50column removing all free peptide (FIGS. 3A-B). Lastly, above experimentswere repeated using previous and newer preparations in the same assay.We extended the two-hour incubation to an overnight period, which hasbeen shown to be sufficient for SIINFEKL (SEQ ID NO: 2) presentation(data not shown). Upon flow cytometric analysis, we observed that thesamples lacking free peptide work just as well as those containingpeptide (FIGS. 3C-E). These results were next confirmed using a B3Zassay (FIG. 3F). Therefore, it is clear that GNPs 8 and 9 serve as aviable option for peptide delivery to an antigen presenting cell forpresentation of MHCI.

D. Conclusions

-   -   GNPs 8, 9, 12, and 13 are processed and presented very well        compared to the others.    -   This corresponds to the sequences FLAAYSIINFEKL (SEQ ID NO: 4)        and AAYSIINFEKL (SEQ ID NO: 5) being the best for in vitro        SIINFEKL (SEQ ID NO: 2) presentation (peptide portion of the        linker shown underlined).    -   HS—(CH2)10-(CH2OCH2)7-CONH chemistry is better processed than        HS(CH2)2-CONH. However, HS(CH2)2-CONH is also processed very        well while being more cost effective.    -   Contaminating free peptide may give rise to presentation.    -   Newer preparations, which lack free peptide, are also processed        and presented well.    -   This suggests that GNPs 8 and 9 are efficiently processed for        presentation of SIINFEKL (SEQ ID NO: 2).

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety.

The specific embodiments described herein are offered by way of example,not by way of limitation. Any sub-titles herein are included forconvenience only, and are not to be construed as limiting the disclosurein any way.

The invention claimed is:
 1. A nanoparticle comprising: (i) a corecomprising a metal and/or a semiconductor atom; (ii) a corona comprisinga plurality of ligands covalently linked to the core, wherein at least afirst ligand of said plurality comprises a carbohydrate moiety that iscovalently linked to the core via a first linker or comprisesglutathione, and wherein at least a second ligand of said pluralitycomprises a peptide of choice that is covalently linked to the core viaa second linker, said second linker comprising: a peptide portion and anon-peptide portion, wherein said peptide portion of said second linkercomprises the sequence FLAAY (SEQ ID NO: 1).
 2. The nanoparticle ofclaim 1, wherein said peptide of choice consists of a sequence of 8 to12 amino acid residues and is an epitopic peptide that binds to a classI Major Histocompatibility Complex (MHC) molecule.
 3. The nanoparticleaccording to claim 1, wherein said non-peptide portion of the secondlinker comprises C2-C15 alkyl and/or C2-C15 glycol.
 4. The nanoparticleaccording to claim 1, wherein said first ligand and/or said secondligand are covalently linked to the core via a sulphur-containing group,an amino-containing group, a phosphate-containing group or anoxygen-containing group.
 5. The nanoparticle according to claim 1,wherein the peptide of choice is an epitopic peptide that is presentedby a class I MHC molecule so as to stimulate a Cytotoxic T Lymphocyte(CTL) response.
 6. The nanoparticle according to claim 1, wherein thepeptide of choice forms at least a portion of or is derived from aTumour-Associated Antigen (TAA) or a viral-, bacterial-, orparasite-associated antigen.
 7. The nanoparticle according to claim 6,wherein the TAA is a lung cancer antigen.
 8. The nanoparticle accordingto claim 1 wherein: (i) the carbohydrate moiety of said first ligandcomprises a monosaccharide and/or a disaccharide; and/or (ii) saidplurality of ligands comprises at least one glutathione ligandcovalently linked to the core of the nanoparticle via the glutathionesulphur atom; and/or (iii) said plurality of ligands comprises: (a)glucose; (b) N-acetylglucosamine; (c) glutathione; (d) glucose andN-acetylglucosamine; (e) glucose and glutathione; (f)N-acetylglucosamine and glutathionie; or (g) glucose,N-acetylglucosamine and glutathione.
 9. The nanoparticle according toclaim 1, wherein said first linker comprises C2-C15 alkyl and/or C2-C15glycol.
 10. The nanoparticle according to claim 1, wherein said firstligand comprises 2′-thioethyl-β-D-glucopyranoside or2′-thioethyl-α-D-glucopyranoside covalently attached to the core via thethiol sulphur atom.
 11. The nanoparticle according to claim 1, whereinthe plurality of ligands are in a molar ratio of carbohydrate-containingligands and/or glutathione ligands to peptide of choice containingligands in the range of 5:1 to 100:1.
 12. The nanoparticle according toclaim 1, wherein the diameter of the core of the nanoparticle is in therange 1 nm to 5 nm.
 13. The nanoparticle according to claim 1, whereinthe core comprises a metal selected from the group consisting of: Au,Ag, Cu, Pt, Pd, Fe, Co, Gd, Zn and any combination of said metals.
 14. Acomposition comprising a plurality of nanoparticles as defined in claim1, and at least one pharmaceutically acceptable carrier, salt and/ordiluents.
 15. The composition according to claim 14, wherein thecomposition comprises a first species of nanoparticle having a firstpeptide of choice-containing ligand and a second species of nanoparticlehaving a second peptide of choice-containing ligand, wherein thepeptides of choice of said first and second species differ.
 16. Thecomposition according to claim 14, further comprising at least oneadjuvant covalently attached to the core of at least one nanoparticle.17. The composition according to claim 16, wherein the adjuvantcomprises(S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys₄-OH(Pam₃Cys).
 18. A vaccine comprising a composition as defined in claim14.